Short range cell-cell signals and inheritance of cytoplasmic determinants provide the information to specify the three germ layers of embryos. This specification occurs early in cleavage in most embryos but the mechanisms by which the germ layers are established are still in the early stages of discovery. In the sea urchin embryo we have learned that beta-catenin is involved in specification of mesoderm and endoderm. Notch is later necessary for specification of secondary mesoderm cells. This proposal is for studies to learn how these sequential specification events work. There are two specific aims in this proposal: Aim 1: To test the hypothesis that nuclear beta-catenin regulates autonomous specification of cells in the vegetal hemisphere during cleavage. All the preliminary evidence supports this hypothesis, however much remains to be learned. How the beta-catenin is activated to enter nuclei and engage in its signaling role is not known. How the required nuclear entry relates to micromere specification and to the known activation of a micromere signal is not known. In macromeres beta-catenin enters nuclei autonomously at the 32-cell stage and these cells also receive a signal from micromeres. The distinct roles of the two signals are not known. Experiments in this section will provide a more complete understanding of how beta-catenin functions autonomously in embryos to activate early specification events. Aim 2: To test the hypothesis that Notch acts after beta-catenin to refine the territories in the vegetal hemisphere and to be essential for secondary mesoderm specification. Again, the preliminary data support this hypothesis and provide a number of clues as to how Notch might work in this early germ layer specification role. We will ask how the earlier beta-catenin signal is essential for the later Notch signal in the vegetal hemisphere. The Notch signal is localized and during signaling the molecule is consumed and degraded. Experiments will explore these molecular observations to better understand the mechanism of Notch's function during this mesoderm specification event. An important component of these experiments is to learn how ligands of Notch and regulators of Notch signaling delineate territorial signaling specificity. In addition, we will experimentally challenge a potential cause and effect relationship between Notch and possible downstream targets of Notch signaling, one of which appears to be activation of the Brachyury mesodermal transcription factor.